1998 Fiscal Year Final Research Report Summary
Transcriptional regulation of cholesterol metabolism
| Project/Area Number |
09557076
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
内分泌・代謝学
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| Research Institution | Research Center for Advanced Science and Technology University of Tokyo |
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Principal Investigator |
KODAMA Tatsuhiko Research Center for Advanced Science and Technology, University of Tokyo, Chair Professor, 先端科学技術研究センター, 教授 (90170266)
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| Co-Investigator(Kenkyū-buntansha) |
KAWAVE Yoshiki Fuji Gotemba Research Labs.Chugai Pharmaceutical CO., LTD,Manager, 創薬研究所, 研究員
HAMAKUBO Takao Research Center for Advanced Science and Technology, University of Tokyo, Lectur, 先端科学技術研究センター, 講師 (90198797)
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| Project Period (FY) |
1997 – 1998
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| Keywords | SREBP / protease / endoplasmic reticulum / cholesterol / cathepsin B / neprilysin / コレステロール / 脂肪酸代謝 |
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| Research Abstract |
Sterol regulatory element binding proteins (SREBP-1 and SREBP-2) are the key transcriptional factors for the feedback regulation of cellular cholesterol level. SREBPs exist as precursor proteins in endoplasmic reticulum(ER)and nuclear membrane. To investigate proteolytic enzymes for SREBPs, a fluorogenic peptide substrate, MOCAc-GRSVLSFK(Dnp)rr-NH_2, was synthesized according to the proposed cleaving site of human SREBP-2. In microsomal fraction from hamster liver, we found dose dependent endopeptidase activity which was inhibited by synthetic inhibitor Ac-GRSVL-aldehyde with an IC_<50> of 4OnM.After solubilization with 0.5% MEGA9, the activities were separated into three peaks at around 400 kda, 60 kda, 30 kda (named Mp400, Mp60 and Mp30 respectively) by gel permeation chromatography. Among these activities, only Mp30 was susceptible to Ac-GRSVL-aldehyde. Following several column chromatographies, Mp30 was purified to apparent homogeneity on SDS-PAGE with an Mr of 32,000. The partial amino acid sequence of Mp30 showed homology to that of single chain cathepsin B (EC 3.4.22.1). A 109 kda protein band on SDS-PAGE which corresponds to Mp400 activity had homology to neprilysin(EC 3.4.24.11)in partial amino acid sequence. These findings suggest the existence of several degradative pathways for ER proteins in liver microsome membranes.
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